Real-time measurement of the intracellular pH of yeast cells during glucose metabolism using ratiometric fluorescent nanosensors

Abstract

Intracellular pH is a key parameter that influences many biochemical and metabolic pathways that can also be used as an indirect marker to monitor metabolic and intracellular processes. Herein, we utilise ratiometric fluorescent pH-sensitive nanosensors with an extended dynamic pH range to measure the intracellular pH of yeast (Saccharomyces cerevisiae) during glucose metabolism in real-time. Ratiometric fluorescent pH-sensitive nanosensors consisting of a polyacrylamide nanoparticle matrix covalently linked to two pH-sensitive fluorophores, Oregon green (OG) and 5(6)carboxyfluorescein (FAM), and a reference pH-insensitive fluorophore, 5(6)carboxytetramethylrhodamine (TAMRA), were synthesised. Nanosensors were functionalised with acrylamidopropyltrimethyl ammonium hydrochloride (ACTA) to confer a positive charge to the nanoparticle surfaces that facilitated nanosensor delivery to yeast cells, negating the need to use stress inducing techniques. The results showed that under glucose-starved conditions the intracellular pH of yeast population (n ≈ 200) was 4.67 ± 0.15. Upon addition of d-(+)-glucose (10 mM), this pH value decreased to pH 3.86 ± 0.13 over a period of 10 minutes followed by a gradual rise to a maximal pH of 5.21 ± 0.26, 25 minutes after glucose addition. 45 minutes after the addition of glucose, the intracellular pH of yeast cells returned to that of the glucose starved conditions. This study advances our understanding of the interplay between glucose metabolism and pH regulation in yeast cells, and indicates that the intracellular pH homestasis in yeast is highly regulated and demonstrates the utility of nanosensors for real-time intracellular pH measurements.

Augmenting Automated Analytics Using Fluorescent Nanosensors

“…When fluorescent nanosensors are delivered to subcellular spaces, they permit direct and indirect quantification of biochemical parameters that could be used to for real-time optimization of growth parameters. Direct measurement of biochemical processes is evidenced by measurement of pH in Saccharomyces cerevisiae [81], whereas indirect measurement of has been shown by the quantification of hydrogen peroxide in human mesenchymal stem cells [82], a toxic by-product of porphyrin induced photodynamic light therapy.…”

Intracellular processing of silica-coated superparamagnetic iron nanoparticles in human mesenchymal stem cells

“…Polyacrylamide-based fluorescent nanosensors are an example of a smart measurement system that allow for complex sensory data to be acquired with minimal sample interference 21. They are spherical particles, ∼50 nm in diameter, which allow for many particles to be delivered to intracellular spaces and provide a high signal-to-noise ratio 22.…”

“…When these fluoresceins are combined with the pH-insensitive reference 5-(and-6)-carboxytetramethylrhodamine to produce fluorescent extended dynamic range pH-sensitive nanosensors, they can be used to make accurate ratiometric measurements that are independent of fluorophore concentration, fluctuations in excitation energy, as well as detector sensitivity and light scattering 26. For example, extended dynamic range pH-sensitive nanosensors have been shown to be capable of making accurate ratiometric measurements to a high spatial (<50 nm), temporal (<100 ms) and pH resolution (±0.17 pH units) in the model organisms Caenorhabditis elegans ,27 Pristionchus pacificus 28 and Saccharomyces cerevisiae 21…”

Enhancing cell and gene therapy manufacture through the application of advanced fluorescent optical sensors (Review)

“…52 Internally, sensors consist of a ratiometric reference dye, which facilitates both tracking of the sensor and ratiometric measurements. 53 Additionally, there must be a sensor dye which reacts to the analyte of interest.…”